Emulsions are typically systems containing at least two immiscible (or substantially immiscible) liquids. Generally, at least one liquid serves as a dispersion phase, the phase dispersed as droplets; whereas at least one other liquid serves as the dispersion medium which is the phase that the droplets are dispersed in. Typical examples include water-in-oil or oil-in-water emulsions. An emulsion is typically a thermodynamically unstable mixture that tends to stay emulsified for a limited time. For example, coalescence occurs when droplets form together into larger droplets and generally indicates increased instability of an emulsion. Creaming occurs when one of the liquids migrates to the top of the emulsion (depending on density), and can sometimes look milky or creamy in appearance.
Emulsions are widely used in biological applications involving nucleic acid manipulation. For example, water-in-oil (w/o) emulsions including a continuous phase of water-immiscible liquid (e.g., oils, organic solvents) in which a discontinuous aqueous phase is dispersed are well known in the field of emulsion PCR. Emulsion PCR generally uses a water-in-oil emulsion, where the oil serves as the dispersion medium while the aqueous phase serves as the dispersion phase. During emulsion PCR, different biomolecules, e.g., proteins or nucleic acid templates, can be individually isolated within the aqueous droplets of the emulsion, with the oil phase acting as a barrier that physically compartmentalizes the templates from each other. Following the desired manipulation (e.g., amplification, expression, cleavage, etc), it is frequently desirable to disrupt or “break” the emulsion to recover the biomolecules from the emulsion. Classical methods of breaking such water-in-oil emulsions including biomolecules in the aqueous phase involve repeated rounds of extraction with organic solvents, such as water-saturated ether. See, e.g., Dressman et al., “Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variation” Proc. Natl. Acad. Sci. 100(15):8817-8822 (2003); Ghadessy et al., “Directed evolution of polymerase function by compartmentalized self-replication”, Proc. Natl. Acad. Sci. 98(8):4552-4557 (2000); Tawfik & Griffiths, “Man-made cell-like compartments for molecular evolution” Nat. Biotech. 16(7):652-656 (1998); Williams et al., “Amplification of complex gene libraries by emulsion PCR” Nat. Meth. 3(7):545-550 (2006). Such methods can be time-consuming, labor-intensive and costly.
Several biological applications also involve manipulation of nucleic acid molecules attached to supports. For example, several next-generation sequencing methods involve amplification and/or analysis of nucleic acid libraries, where individual members of the libraries are attached to particles. For such applications, it can be useful to obtain relatively accurate measure of the number of particles within a sample, as well as of the number of particles that are attached to particular nucleic acid sequences. For example, in methods involving extension of primers attached to particles, it can be useful to measure the number of particles including extended primers attached thereto (thereby gaining an indication of the efficiency of the primer extension reaction). Traditional methods of analyzing nucleic acid populations attached to particles involve individual assessment of one or more particles. For example, particle concentration is frequently estimated by counting of individual particles via flow cytometry, a costly and time consuming process. Similarly, the amount of particles including extended primers is typically assessed via hybridization of the particles to sequence-specific probes that hybridize selectively to extended portions of the primer, and visualizing such particles under the microscope to determine how many particles are hybridized to the sequence-specific probe. There is therefore a need for improved methods, compositions and systems that allow rapid, simple and inexpensive breaking of water-in-oil emulsions including biomolecules dispersed in the aqueous phase without significantly denaturing or otherwise disrupting the biomolecules or impairing their ability to participate in downstream manipulations such as nucleic acid sequencing, enzymatic reactions and the like.
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